Process for the preparation of ethyl 3S-[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate

ABSTRACT

The present invention relates to a novel process for the preparation of ethyl 3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate and pharmaceutically acceptable acid addition salt thereof which comprises treating (trimethylsilyl)acetylene sequentially with n-butyllithium and 4-formylmorpholine followed by acid hydrolysis to give 3-(trimethylsilyl)-2-propynal; treating 3-(trimethylsilyl)-2-propynal with lithium bis(trimethylsilyl)amide to give in situ N,3-bis(trimethylsilyl)-2-propyn-1-imine; condensation of N,3-bis(trimethylsilyl)-2-propyn-1-imine with lithium t-butyl acetate to give (±)1,1-dimethylethyl 3-amino-5-(trimethylsilyl)-4-pentynoate; treating (±)1,1-dimethylethyl 3-amino-5-(trimethylsilyl)-4-pentynoate- with p-toluenesulfonic acid to give (±)1,1-dimethylethyl 3-amino-5-(trimethylsilyl)-4-pentynoate, mono p-toluenesulfonic acid salt, treatment of resulting salt with ethanol in the presence of p-toluenesulfonic acid to give (±)ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate; desilylation of (±)ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate to give in situ (±)ethyl 3-amino-4-pentynoate; resolution of (±)ethyl 3-amino-4-pentynoate using (R)-(-)-mandelic acid and treatment of the resolved product with gaseous hydrochloric acid to give ethyl 3S-amino-4-pentynoate, monohydrochloride; coupling the ethyl 3S-amino-4-pentynoate, monohydrochloride to 4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid, monohydrochloride in the presence of isobutyl chloroformate and N-methylmorpholine to give ethyl 3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate, monohydrochloride.

BACKGROUND OF THE INVENTION

The present invention relates to a novel process for the preparation ofethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoatehaving the following structural formula ##STR1## and thepharmaceutically acceptable acid addition salt thereof which comprisestreating (trimethylsilyl) acetylene sequentially with n-butyllithium and4-formylmorpholine in the presence of an aprotic solvent followed byacid hydrolysis to give 3-(trimethylsilyl)-2-propynal; treating3-(trimethylsilyl)-2-propynal with lithium bis(trimethylsilyl)amide inthe presence of an aprotic solvent to giveN,3-bis(trimethylsilyl)-2-propyn-1-imine in situ; condensation ofN,3-bis(trimethylsilyl)-2-propyn-1-imine with lithium t-butyl acetatefollowed by hydrolytic cleavage to give (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate; treating (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate with p-toluenesulfonic acid inthe presence of aprotic solvents to give (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate, mono p-toluenesulfonic acidsalt; treatment of the resulting salt with ethanol in the presence ofp-toluenesulfonic acid, followed by neutralization to give (±)ethyl3-amino-5-(trimethylsilyl)-4-pentynoate; desilylation of (±)ethyl3-amino-5-(trimethylsilyl)-4-pentynoate in the presence of a catalyticamount of base and an alkanol solvent to give in situ (±)ethyl3-amino-4-pentynoate; resolution of (±)ethyl 3-amino-4-pentynoate using(R)-(-)-mandelic acid and treatment of the resolved product with gaseoushydrochloric acid to give ethyl 3S-amino-4-pentynoate,monohydrochloride; coupling the ethyl 3S-amino-4-pentynoate,monohydrochloride to 4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoicacid, monohydrochloride in the presence of isobutyl chloroformate andN-methylmorpholine to give ethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate,monohydrochloride.

The 4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid,monohydrochloride intermediate is prepared by treating commerciallyavailable 4-aminobenzamidine dihydrochloride with succinic anhydride andpyridine in the presence of an aprotic solvent.

The process of the present invention is useful for preparing ethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoatewhich is described in U.S. Pat. No. 5,344,957.

Ethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoateis the orally active prodrug of3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoicacid. The acid form is an active platelet aggregation inhibitor. Acomplete discussion of ethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoateand3S-[[4-[[4-(aminoiminomethyl)-phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoicacid usefulness as a platelet aggregation inhibitor is presented in theU.S. Pat. No. 5,344,957 patent.

U.S. Pat. No. 5,344,957 discloses a process for preparing ethyl 3S3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoatehaving the following formula ##STR2## This process is described asfollows: 4-Aminobenzamidine di-HCl (25 g, 120 mmol), which iscommercially available, particularly from Aldrich, was added to dry DMF(100 ml). To this solution dry pyridine (100 ml) and succinic anhydride(12 g, 120 mmol) followed by dimethylaminopyridine (DMAP 1.5 g 0.012mmol) were added. The product precipitated after heating for 1/2 h at100° C. The product was filtered, washed with water, acetonitrile andether. The light solid was suspended in dioxane, 4N HCl in dioxane (100ml) was added and the suspension was stirred for 1 h, filtered and driedin a desiccator to give 28 g, 88% of4-[[4-(aminoiminomethyl)phenyl]-amino]-4-oxobutanoic acid as a whiteyellow solid which decomposes between 270° C. and 290° C.

4-[[4-(Aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid hydrochloridewas added to dry DMF (35 ml) followed by N-methylmorpholine (0.39 g, 1eq.) and isobutyl chloroformate (0.53 g, 3.9 mmol) at 25° C. The mixturewas stirred for 5 min. (S)-ethyl 3-amino-4-pentynoate was added followedby diisopropylethylamine and a catalytic amount ofdimethylaminopyridine. After 1 hour, the solvent was removed underreduced pressure and the product was purified by reverse phasechromatography to give (3S)-ethyl3-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate.

The (S)ethyl 3-amino-4-pentynoate intermediate which is described abovewas prepared according to the procedures disclosed in Method 3 of SchemeV of the "957" patent. Similar reactions are also disclosed by D. H. Huaand A. Verma, Tetrahedron Lett. 547-550 (1985) and T. Kametani,Heterocycles Vol. 17 463 (1982). These references are also disclosed inthe "957" patent.

SUMMARY OF THE INVENTION

The present invention relates to a process for the preparation of ethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoatehaving the following formula: ##STR3## and the pharmaceuticallyacceptable salt thereof which comprises: (a) treating(trimethylsilyl)acetylene sequentially with n-butyllithium and4-formylmorpholine in the presence of an aprotic solvent followed byacid hydrolysis to give 3-(trimethylsilyl)-2-propynal;

(b) treating 3-(trimethylsilyl)-2-propynal, the product of step a, withlithium bis(trimethylsilyl)amide in the presence of an aprotic solventto give N,3-bis(trimethylsilyl)-2-propyn-1-imine in situ, treatingN,3-bis(trimethylsilyl)-2-propyn-1-imine with lithium t-butyl acetatefollowed by hydrolytic cleavage to give (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate;

(c) treating (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate, the product of step b, withp-toluenesulfonic acid in the presence of aprotic solvents to give(±)1,1-dimethylethyl 3-amino-5-(trimethylsilyl)-4-pentynoate, monop-toluenesulfonic acid salt, treating the resulting salt with ethanol inthe presence of p-toluenesulfonic acid, followed by neutralization togive (±)ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate;

(d) treating (±)ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate, theproduct of step c, with a catalytic amount of base in the presence of analkanol solvent followed by a catalytic amount of acid to give thedesilylated (±)ethyl 3-amino-4-pentynoate in situ, treating (±)ethyl3-amino-4-pentynoate with (R)-(-)-mandelic acid in the presence ofaprotic solvents to give ethyl 3S-amino-4-pentynoate compounded withαR-hydroxybenzeneacetic acid;

(e) treating 4-aminobenzamidine dihydrochloride with succinic anhydrideand pyridine in the presence of an aprotic solvent to give4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid,monohydrochloride;

(f) treating ethyl 3S-amino-4-pentynoate compounded withαR-hydroxybenzeneacetic acid, the product of step d, with gaseoushydrochloric acid in the presence of an aprotic solvent to give ethyl3S-amino-4-pentynoate, monohydrochloride; and

(g) treating 4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid,monohydrochloride, the product of step e, with isobutyl chloroformateand N-methylmorpholine in the presence of an aprotic solvent followed byethyl 3S-amino-4-pentynoate, monohydrochloride, the product of step f,with N-methylmorpholine to give ethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate,monohydrochloride with the understanding that when a pharmaceuticallyacceptable acid addition salt other than hydrochloride is desired theethyl 3S-amino-4-pentynoate compounded with αR-hydroxybenzeneaceticacid, the product of step d, is treated with the appropriate acidcorresponding to the desired salt and with the further understandingthat the final salt of step e is identical to the final salt of step f.

The present invention also relates to a process for preparing ethyl3S-amino-4-pentynoate monohydrochloride having the following formula##STR4## which comprises: (a) treating (trimethylsilyl)acetylenesequentially with n-butyllithium and 4-formylmorpholine in the presenceof an aprotic solvent followed by acid hydrolysis to give3-(trimethylsilyl)-2-propynal;

(b) treating 3-(trimethylsilyl)-2-propynal, the product of step a, withlithium bis(trimethylsilyl)amide in the presence of an aprotic solventto give N,3-bis(trimethylsilyl)-2-propyn-1-imine in situ, treatingN,3-bis(trimethylsilyl)-2-propyn-1-imine with lithium t-butyl acetatefollowed by hydrolytic cleavage to give (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate;

(c) treating (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate, the product of step b, withp-toluenesulfonic acid in the presence of aprotic solvents to give(±)1,1-dimethylethyl 3-amino-5-(trimethylsilyl)-4-pentynoate, monop-toluenesulfonic acid salt, treating the resulting salt with ethanol inthe presence of p-toluenesulfonic acid, followed by neutralization togive (±)ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate; and

(d) treating (±)ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate, theproduct of step c, with a catalytic amount of base in the presence ofalkanol solvent followed by a catalytic amount of acid to give thedesilylated (±)ethyl 3-amino-4-pentynoate in situ, treating (±)ethyl3-amino-4-pentynoate with (R)-(-)-mandelic acid in the presence ofaprotic solvents to give ethyl 3S-amino-4-pentynoate compounded withαR-hydroxybenzeneacetic acid; and

(e) treating ethyl 3S-amino-4-pentynoate compounded withαR-hydroxybenzeneacetic acid, the product of step d, with gaseoushydrochloric acid in the presence of an aprotic solvent to give ethyl3S-amino-4-pentynoate, monohydrochloride; with the understanding thatwhen a pharmaceutically acceptable acid addition salt other thanhydrochloride is desired the ethyl 3S-amino-4-pentynoate compounded withαR-hydroxybenzene acetic acid, the product of step d, is treated withthe appropriate acid corresponding to the desired salt. ##STR5##

The aforementioned chemical names have the following structuralformulas: ##STR6##

As used herein the term "pharmaceutically acceptable acid addition salt"refers to a salt prepared by contacting a compound of formula I, with anacid whose anion is generally considered suitable for human consumption.Examples of pharmacologically acceptable salts include thehydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate,propionate, lactate, maleate, malate, succinate, and tartrate andcitrate salts.

DETAILED DESCRIPTION OF THE INVENTION

The starting material for the process of thisinvention--(trimethylsilyl)acetylene--is commercially available. Theprocess of the present invention is outlined in Scheme A and isdescribed as follows:

Step 1 of Scheme A

The formylation step (step 1) of Scheme A is carried out at atemperature ranging from -45° to +5° C. with -10° C. being preferredusing n-butyllithium in hexanes in the presence of an aprotic solventand (trimethylsilyl) acetylene as the protected alkyne. To this reactionmixture with stirring at a temperature ranging from -15° to 5° C. isadded 4-formylmorpholine. The resulting reaction mixture is allowed tostir for 2 h at a temperature ranging from 0° to 25° C., cooled to atemperature ranging from -15° to 0° C. and then added to an aqueoussolution of HCl. This step yields 3-(trimethylsilyl)-2-propynal which isisolated from the reaction mixture and used in the next step. Suitableaprotic solvents are exemplified by tetrahydrofuran, dioxane, ethylacetate and lower alkyl ethers which do not react with n-butyllithiumsuch as methyl t-butyl ether (MTBE) with methyl t-butyl ether beingpreferred.

Step 2 of Scheme A

Lithium bis(trimethylsilyl)amide is placed in a reaction vessel to which3-(trimethylsilyl)-2-propynal of step 1 is added in an aprotic solvent.During the addition of 3-(trimethylsilyl)-2-propynal the pot temperatureshould be maintained between -40° to -20° C. to ensure stability of theintermediate imine. To the resulting solution is added trimethylsilylchloride and the resulting mixture stirred at a temperature ranging from-40° to -20° C. for 1 hour. This reaction step yieldsN,3-bis(trimethylsilyl)-2-propyn-1-imine which is not isolated from thevessel but is confirmed by GC. To the reaction vessel is now addedt-butyl acetate and a catalytic amount of lithiumbis(trimethylsilyl)amide. The reaction mixture is allowed to stir for 2h at a temperature ranging from -40° to -20° C. to give(±)1,1-dimethylethyl 3-amino-5-(trimethylsilyl)-4-pentynoate which isisolated after an aqueous ammonium chloride quench and used in the nextstep. In order to insure complete cleavage of the nitrogen-silicon bondthe stir time for this phase should not be less than 30 minutes.

Step 3 of Scheme A

The p-toluenesulfonic acid salt of (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate is prepared by treatingp-toluenesulfonic acid with (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate, the product of step 2, in thepresence of MTBE/heptane. The resulting reaction mixture is then heatedto 55°-60° C. for one hour, cooled, filtered and dried to give (±)1,1,-dimethylethyl 3-amino-5-(trimethylsilyl)-4-pentynoate, monop-toluenesulfonic acid salt. The (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate mono p-toluenesulfonic acid saltis treated with ethanol in the presence an organic acid or an inorganicacid at reflux temperature. The reaction mixture is then cooled toambient temperature, concentrated and treated with methyl t-butyl etherand a suitable aqueous base to give ethyl3-amino-5-(trimethylsilyl)-4-pentynoate. Suitable inorganic acid agentsare exemplified by sulfuric acid and hydrochloric acid and suitableorganic acid agents are exemplified by camphorsulfonic acid andp-toluenesulfonic acid with p-toluenesulfonic acid being preferred.Suitable aqueous bases are exemplified by sodium bicarbonate, sodiumcarbonate, sodium hydroxide with sodium bicarbonate being preferred.Other alkanol solvents such as methanol, isopropanol, butanol andpentanol would also be suitable for the practice of this invention.However, it is to be understood that the use of a different alkanolsolvent would give a different ester i.e. use of methanol for ethanolwould give the corresponding methyl ester.

Step 4 of Scheme A

(±) Ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate the product of step 3is desilylated in the presence of a catalytic amount of base and analkanol solvent. Suitable bases are exemplified by potassiumtert-butoxide, sodium hydroxide, sodium ethoxide and sodium carbonatewith sodium ethoxide being preferred. The basic reaction is neutralizedwith an inorganic or organic acid to give an alkanol solution of ethyl3-amino-4-pentynoate which is used in the next step without isolation.Suitable inorganic acid agents are exemplified by hydrochloric acid,sulfuric acid and phosphoric acid with suitable organic agents beingexemplified by p-toluenesulfonic acid, acetic acid, camphorsulfonic acidand cationic exchange resin with sulfuric acid being preferred. Suitablealkanol solvents are exemplified by methanol, ethanol, isopropanol andbutanol with ethanol being preferred. Resolution of (±) ethyl3-amino-5-(trimethylsily)-4-pentynoate using R-(-)-mandelic acid in thepresence of aprotic solvents gives ethyl 3S-amino-4-pentynoatecompounded with αR-hydroxybenzeneacetic acid. Suitable aprotic solventsare exemplified by tetrahydrofuran, dioxane, ethyl acetate, and loweralkyl ethers such as methyl t-butyl ether with a mixture of ethylacetate and methyl t-butyl ether being preferred.

Step 5 of Scheme A

Treatment of 4-aminobenzamidine dihydrochloride, which is commerciallyavailable, with succinic anhydride in the presence ofN,N-dimethylformamide and pyridine gives4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid, as a mixtureof the zwitterion and the hydrochloride. The product is isolated fromthe crude reaction mixture by the addition of conc HCl in acetonefollowed by additional acetone. Filtration and treatment with aqueousHCl gives 4-[[4-(aminoiminomethyl)phenyl]-amino]-4-oxobutanoic,monohydrochloride.

Step 6 of Scheme A

Treatment of ethyl 3S-amino-4-pentynoate compounded withαR-hydroxybenzeneacetic acid (the product of step 4) with gaseoushydrochloric acid in the presence of an aprotic solvent gives ethyl3S-amino-4-pentynoate, monohydrochloride. Suitable aprotic solvents areexemplified by tetrahydrofuran, dioxane, ethyl acetate and lower alkylethers such as methyl t-butyl ether (MTBE) with methyl t-butyl etherbeing preferred.

Step 7 of Scheme A

4-[[4-Aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid,monohydrochloride, the product of Step 5, is coupled with ethyl3S-amino-4-pentynoate hydrochloride, the product of Step 6, in presenceof isobutyl chloroformate and N-methylmorpholine to give ethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate,monohydrochloride. The reaction temperature for this step should bemaintained between -15° to 0° C. The reaction sequence is the following:

(a) the product of step 5 which is4-[[4-aminoiminomethyl)phenyl]amino]-4-oxobutanoic, hydrochloride inN,N-dimethylacetamide (DMAc) is cooled to -15° to -10° C.;

(b) to the cooled reaction mixture is added isobutyl chloroformate(IBCF) followed by N-methylmorpholine (NMM);

(c) to this reaction mixture is now added ethyl 3S-amino-4-pentynoatehydrochloride in DMAc, followed by the addition of N-methylmorpholine;and

(d) the resulting reaction mixture is warmed to ambient temperature andthe desired product isolated. Suitable solvents in place of thepreferred N,N-dimethylacetamide (DMAc) would be N,N-dimethylformamide(DMF) and N-methylpyrrolidinone (NMP).

The present invention provides a safe, convenient and cost effectivemanufacturing process for the production of ethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4dioxobutyl]amino-4-pentynoate, monohydrochloride. Its safety isdemonstrated by the elimination of potentially hazardous solvents andreagents. The subject process in step 5 of Scheme A utilizes aqueousHCl/acetone mixture in place of the ether and dioxane of the "957"patent. Its convenience is demonstrated by the synthetic routecomprising a limited number of steps. Its cost effectiveness isdemonstrated by the final product being produced in high yield and highquality.

A major difference between the process described in the "957" patent forthe preparation ethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate,monohydrochloride and the process of the present invention is in the useof a different solvent, a lower reaction temperature and the absence of4-dimethylaminopyridine during the coupling reaction as well as theelimination of chromatography and lyophilization. In "957" patent, thereaction is conducted in DMF at 25° C. for both the activation andcoupling steps. In addition, 4-dimethylaminopyridine is added during thecoupling phase.

The following examples are provided to illustrate the present inventionand are not intended to limit the scope thereof. All temperaturesexpressed are in degrees centigrade. Within the foregoing syntheticdescription and examples which follow, abbreviations have the followingmeanings:

ACN=acetonitrile

DMF=N,N dimethylformamide

DMSO=dimethylsulfoxide

g=gram

EtOH=ethanol

Ether=diethylether

min=minute

h=hour

mL=milliliter

mol=mole

mmol=mmole

NMM=N-methylmorpholine

RPHPLC=Reverse Phase High Pressure Liquid Chromatography

LiHMDS=lithium bis(trimethylsilyl)amide

THF=tetrahydrofuran

TMSCl=trimethylsilyl chloride

NaOEt=sodium ethoxide

p-TsOH=p-toluenesulfonic acid

MTBE=methyl t-butyl ether

IBCF=isobutyl chloroformate

DMAc=N,N-dimethylacetamide

Infrared (IR) spectra are recorded on a Perkin-Elmer® 681spectrophotometer. Nuclear magnetic resonance (NMR) spectra are obtainedon a Varian® VXR-400 spectrometer using tetramethylsilane as internalstandard. Elemental analyses are obtained using a Control EquipmentModel 240XA Elemental Analyzer (C, H and N) and a Mettler potentiometrictitration system (total Cl and Cl). Differential scanning calorimetry(DSC) analyses are obtained using a DuPont® Model 9900 thermal analysissystem.

Chemical reactions are monitored by gas chromatography (GC), HighPressure Liquid Chromatography (HPLC) or thin layer chromatography (TLC)on Macherey-Nagel® SIL G-25 UV₂₅₄ silica gel plates. Chemicalintermediates are analyzed by GC or high performance liquidchromatography (HPLC). Qualitative estimates of purity are based uponintegration of the area under the peaks in the GC or HPLC chromatograms.

    ______________________________________                                        GC Method A                                                                   Column      Rtx-5 (5% Diphenyl, 95% dimethyl                                              polysiloxane; 30 m × 0.53 mm i.d.,                                      5-micron film thickness)                                          Oven Temp.  35° C. (2 min) to 250° C. (2 min) at                            10° C./min                                                 Injecter Temp.                                                                            180° C.                                                    Detector Temp.                                                                            250° C.                                                    Detection   FID                                                               Column Flow Rate                                                                          Helium at 4 mL/min                                                Split Mode  Splitless                                                         Injection Volume                                                                          1 mcL                                                             Sample      5 mg/mL in methyl t-butyl ether                                   Concentration                                                                             20-30 mg/mL in methyl t-butyl ether                               ______________________________________                                        GC Method B                                                                   Column      DB-17 (50% Diphenyl, 50% methyl                                               silicone; 15 m × 0.53 mm i.d.,                                          1-micron film thickness)                                          Oven Temp.  35° C. (5 min) to 250° C. (5 min) at                            10° C./min                                                 Injecter Temp.                                                                            250° C.                                                    Detector Temp.                                                                            250° C.                                                    Detection   FID                                                               Column Flow Rate                                                                          Helium at 20 mL/min                                               Split Mode  Splitless                                                         Injection Volume                                                                          1 mcL                                                             Sample      3 mg/mL                                                           Concentration                                                                             1 mg/mL                                                           Derivatization                                                                            N-Methyl-N-(trimethylsilyl)-                                                  trifluoroacetamide (MSTFA)                                        ______________________________________                                        GC Method C                                                                   Column      HP-1 (Methyl silicone; 15 m × 0.53 mm                                   i.d., 2.65-micron film thickness)                                 Oven Temp.  50° C. (5 min) to 250° C. (10 min) at                           15° C./min                                                 Injecter Temp.                                                                            240° C.                                                    Detector Temp.                                                                            260° C.                                                    Detection   FID                                                               Column Flow Rate                                                                          Helium at 20 mL/min                                               Split Mode  Splitless                                                         Injection Volume                                                                          0.5 mcL                                                           Sample      5 mg/mL in methyl t-butyl ether                                   Concentration                                                                             3 mg/mL in methyl t-butyl ether                                   ______________________________________                                        HPLC Method A                                                                 Column      Chiralcel OD (25 cm × 4.6 mm)                               Column Temp.                                                                              Ambient                                                           Mobile Phase                                                                              Hexane/Isopropanol, 85/15 (v/v)                                   Flow Rate   2 mL/min                                                          Sample      1.6 mg/mL                                                         Concentration                                                                 Injection Volume                                                                          10 mcL                                                            Detection   UV at 230 nm                                                      ______________________________________                                        HPLC Method B                                                                 Column      YMC AQ-303 (25 cm × 4.6 mm)                                 Column Temp.                                                                              Ambient                                                           Mobile Phase                                                                              Acetonitrile/1% Triethylammonium                                              phosphate buffer, pH 3, 5/95 (v/v)                                Flow Rate   1 mL/min                                                          Sample      1 mg/mL in mobile phase                                           Concentration                                                                             0.5 mg/mL in mobile phase                                         Injection Volume                                                                          10 mcL                                                            Detection   UV at 210 nm                                                      ______________________________________                                    

EXAMPLE 1 ##STR7##

To a 1 L, three-necked round bottomed flask equipped with a mechanicalstirrer, a pressure-equalized dropping funnel, a temperature probe and anitrogen inlet was charged 59 mL of (trimethylsilyl)acetylene and 210 mLof methyl t-butyl ether (MTBE) at ambient temperature affording a paleyellow homogeneous solution. This homogeneous solution was mechanicallystirred while being cooled with an acetone/dry ice bath to at least -15°C. The dropping funnel was charged with 245 mL of n-butyllithium(n-BuLi) (1.6M; hexanes) which was added as fast as possible to the coldsolution while maintaining the batch temperature below 0° C. Theresulting homogeneous solution was stirred at -15° C. for 10 to 15 min.A new, clean dropping funnel was charged with 40 mL of4-formylmorpholine which was added as fast as possible to the coldsolution while maintaining the batch temperature below 15° C. Theresulting homogeneous solution was then warmed to ambient temperature(ca. 22° C.) by removing the cooling bath. After stirring forapproximately 2 h, during which time the reaction mixture becameheterogeneous, the reaction mixture was cooled in an acetone/dry icebath to -15° C. While the reaction mixture was being cooled again, 340mL of a pre-made 10% aq HCl solution was charged to a 1 L erlenmeyerflask and magnetically stirred at ambient temperature (ca. 22° C.). Oncethe organic solution reached -15° C., the heterogeneous solution wascharged as quickly as possible to 300 mL of the 10% aq HCl solution andthe resulting mixture was magnetically stirred for at least 15 min.During this time, the heterogeneous organic layer changes to atransparent yellow one. An in-process check (IPC) was performed on analiquot using Gas Chromatrography (GC) Method A to confirm the presenceof the desired product. The quenched mixture was charged to a separatoryfunnel. The layers were separated and the organic layer was washed with300 mL of 20% aq. NaCl and the layers were separated. The solvent wasremoved by reduced pressure resulting in 45.7 g of the title compound(molar yield =91%) as a 60-95 wt % solution. The concentration of thedesired product is determined by an acceptance test using GC Method B.An analytical sample may be obtained by reduced pressure distillation.

A sample of purified title compound analyzed as follows:

¹ H NMR (CDCl₃) δ8.90 ppm (s, 1H, CHO), 0.00 (s, 9H, TMS) ppm; ¹³ C NMR(CDCl₃) δ176.5, 102.8, 102.2, 1.0 ppm; IR: 2155 (C.tbd.C), 1667 (CHO)CM⁻¹ ; Anal calc'd for C₆ H₁₀ OSi: C, 57.09; H, 7.98; Found: C, 56.86;H, 8.25.

EXAMPLE 2 ##STR8##

To a 500 mL, 3-neck round bottom flask equipped with a mechanicalstirrer and under a blanket of nitrogen was charged 110 mL of lithiumbis(trimethylsilyl)amide (1.0M solution in THF) while maintaining theinternal temperature at -20° C. using an isopropanol/dry ice bath. Oncethe desired temperature was attained, 20.14 g of3-(trimethylsilyl)-2-propynal (a 62.7% solution in MTBE) was added tothe reaction flask over 2 h. Upon completing the addition, the reactionmixture was stirred for 10 min at -20° C. followed by the addition of10.3 g of trimethyl silyl chloride over a period of 20 min whilemaintaining the batch temperature at -20° C. Upon completing theaddition the reaction mixture was stirred for 10 min at which time 69.7g of t-butyl acetate was added to the reaction mixture over 15 min whilemaintaining the batch temperature at -20° C. The reaction mixture wasstirred for 10 min at -20° C. at which time 19.2 mL of lithiumbis(trimethylsilyl)amide (1.0M solution in THF) was added over a periodof 40 min while maintaining the batch temperature at -20° C. Anin-process check was performed on an aliquot using GC Method C to ensurethat the area % of the desired product was >55%. The reaction mixturewas added to an ammonium chloride solution (prepared by adding 5.0 g ofammonium chloride in 25 mL of water) and the mixture was allowed toreach ambient temperature with stirring (25° C.). The mixture wasstirred at ambient temperature (25° C.) for 1 h. The mixture wastransferred to a separatory funnel and the phases were separated. Anin-process check was performed using GC Method C to determine thepresence of desired product. This material was used without furtherpurification. The solvent from the organic phase was removed underreduced pressure to give 34.5 g of the title compound as a brown oil(49.52 wt % , a molar yield of 70.7%).

An analytical sample may be obtained by crystallization of thep-toluenesulfonic acid salt from heptane/MTBE. Liberation of the freeamine by treatment with aqueous potassium carbonate and extraction intoan organic solvent such as MTBE gives (following removal of solvent)pure (±)1,1-dimethylethyl 3 amino-5-(trimethylsilyl)-4-pentynoate.Alternatively, the free amine may be purified by distillation undervacuum.

A sample of purified (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate analyzed as follows: ¹ H NMR(CDCl₃, 400 MHz) δ3.96(1H, dd), 2.60-2.47 (2H, m), 1.65 (2H, s, --NH₂),1.45 (9H, s), 0.15 (9H, s). ¹³ C NMR (CDCl₃, 125 MHz) δ170.05, 107.96,86.60, 80.85, 43.87, 41.02, 28.05 (3C), -0.13 (3C). Anal. calcd for C₁₂N₂₃ NO₂ Si: C, 59.70; H, 9.60: N, 5.80. Found: C, 59.85; H, 9.67; N,5.74.

EXAMPLE 3 ##STR9##

A mixture containing 80 g of p-toluenesulfonic acid, 116 mL of MTBE, 829mL of heptane and 280 g of (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate (35.7 wt % solution in THF) washeated to 55°-60° C. for 1 h. The homogeneous solution was cooled to 40°C. over 1 h and then to 5° C. over 1 h at which time the resultingheterogeneous mixture was held at 5° C. for 1 h. The mixture was vacuumfiltered and the cake was washed with 2×160 mL of heptane and dried onthe filter with a nitrogen flow to give 142 g of (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate, mono p-toluenesulfonic acidsalt (83% molar yield) which was used without further purification.

A solution containing 142 g of (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate, mono p-toluenesulfonic acidsalt and 20 g of p-toluenesulfonic acid in 260 mL of 2B ethanol wasstirred and heated to reflux. After refluxing for 4 h, the reactionmixture was cooled to ambient temperature and concentrated on a rotaryevaporator. With vigorous agitation, 344 mL of MTBE was added to thedistillation residue followed by 949 g of 5% aq NaHCO₃ with considerablegas evolution observed during the latter. The mixture was stirred for 30min and the layers were separated. The aqueous layer was extracted with140 mL of MTBE; the layers were separated and the organic phases werecombined. The combined organic phases were washed with 206 g of 7% aqNaHCO₃ and allowed to stand for 15 min before the layers were separated.The organic layer was dried over MgSO₄, filtered, and concentrated onthe rotary evaporator to afford 60.4 g of the title compound (83% molaryield) as a yellow oil: ¹ H NMR (CDCl₃) δ4.19 (q, 2H), 4.0 (m, 1H), 2.6(m, 1H), 1.65 (s, 1H), 1.28 (t, 3H), 0.15 (s, 9H) ppm; ¹³ C NMR: 170.7,107.7, 86.8, 60.5, 42.7, 40.8, 14.1, -0.2 ppm; IR (neat): 3315-3383 cm⁻¹(NH), 2960-2978 cm.sup. -1 (CH), 2105-2166 cm⁻¹ (C.tbd.C) , 1735 cm⁻¹(C═O); Anal. Calcd for C₁₀ H₁₉ NO₂ Si: C, 56.30; H, 8.98; N, 6.56;Found: C, 56.67; H, 8.77; N, 6.27.

EXAMPLE 4 ##STR10## Desilylation of (±)ethyl3-amino-5-(trimethylsilyl)-4-pentynoate

To a 3 L round bottomed flask equipped with a magnetic stirrer, apressure-equalized addition funnel, a thermocouple and nitrogen inletwas added 305.4 g of (±)ethyl 3-amino-5-(trimethylsilyl)-4-pentynoateand 800 mL of 2B ethanol. To the resulting homogeneous solution wasadded dropwise 90.0 g of a 21 wt % solution of sodium ethoxide inethanol. The reaction was stirred at 25° C. for 30 min. To the basicsolution was added 14.1 g (7.8 mL) of concentrated sulfuric acid and theresulting reaction mixture was stirred for 30 min.

Mandelic Acid Salt Formation and Isolation

To the above solution was added 213.3 g of (R)-(-)-mandelic acid all atonce and the mixture was stirred for 60 min at 25° C. The bulk of thesolvent was removed by distillation under reduced pressure maintainingthe batch temperature below 35° C. The resulting residue was suspendedin 500 mL of ethyl acetate and the solvent was once again removed bydistillation under reduced pressure to remove remaining ethanol. Theethyl acetate treatment was repeated two more times. The resulting milkyresidue was suspended in 1.6 L of ethyl acetate which was transferred toa jacketed 2 L non-baffled vessel. To this heterogeneous solution wasadded 450 mL of MTBE and the resulting mixture was warmed to 30° C.resulting in a transparent brown solution. The warmed solution wascooled to 0° C. over 3 h. The crystallization mixture was held at 0° C.for 4 h. The solids were isolated by filtration, washed with 2×200 mL ofethyl acetate:MTBE (30:70 v/v) and dried at 30°-40° C. affording 124 gof a beige solid (30% molar yield). The chirality of the title productwas determined to be 92% S by HPLC Method A.

Recrystallization

To 122 g of the above solids was added 1.3 L of acetonitrile and themixture was stirred 1 h at ambient temperature. To the homogeneoussolution was added 60 g of Solka floc™ filter aid and the resultingheterogeneous mixture was filtered using house vacuum. The filter cakewas washed with 2×100 mL of acetonitrile. The filtrate was concentratedusing a rotary evaporator to a final concentration of 5 L ofacetonitrile per kg of crude ethyl 3S-amino-4-pentynoate compounded withαR-hydroxybenzene acetic acid. The concentration was monitored bymeasuring the weight remaining in the distillation vessel. To theconcentrated solution was added 0.6 L of MTBE (5 L/kg) and theheterogeneous mixture was heated to 35°-50° C. to dissolve the solids.The solution was cooled to 0° C. over 3 h. The crystallization was heldfor at least 4 h at 0° C. The solids were isolated by vacuum filtration,washed with 3×50 mL of MTBE and dried in a vacuum oven at 30°-40° C. toafford 89.4 g ethyl 3S-amino-4-pentynoate compounded withαR-hydroxybenzeneacetic acid (21% molar yield from (±)ethyl3-amino-5-(trimethylsilyl)-4-pentynoate). The chirality of the titleproduct was determined to be 99.6% S by HPLC Method A. DSC endotherm at102.17° C.; IR: (KBr) 3297 cm⁻¹ (N--H), 2132 cm⁻¹ (C.tbd.C), 1744 cm⁻¹(C═O), 1563 cm⁻¹ (C═O); ¹ H NMR: (CDCl₃) δ7.42-7.2 (m, 5H), 6.0 (br.s,4H), 5.0 (s, 1H), 4.15 (q, 2H), 4.03 (m, 1H), 2.67 (m, 2H), 2.32 (d,1H), 1.25 (t, 3H) ppm; ¹³ C NMR (CDCl₃): δ178.4, 170.0, 140.6, 128.3,127.6, 126.7, 79.9, 74.5, 74.1, 61.3, 39.2, 38.5, 14.1 ppm; Anal. calcdfor C₁₅ H₁₉ NO₅ : C, 61.42; H, 6.53; N, 4.78 Found: C, 61.43; H, 6.56;N, 4.63.

Hydrochloride Salt Formation

A suspension of 18.0 g of ethyl 3S-amino-4-pentynoate compounded withαR-hydroxybenzeneacetic acid and 178 mL of MTBE were stirred at ambienttemperature for 15 min at which time the suspension was cooled to 10° C.in an ice/water bath. Gaseous HCl (5.2 g) was slowly bubbled into thereaction mixture while maintaining the batch temperature at 10° C. Theice bath was removed allowing the reaction to warm to ambienttemperature. After 4.5 h, the reaction mixture was filtered, the solidswere washed with MTBE and air dried overnight to afford 9.9 g of thetitle compound (94% molar yield):DSC:endotherm at 133.67° C., ¹ H NMR(C₂ D₅ OD) δ5.21 (br.s, 3H), 4.45 (m, 1H), 4.2 (q, 2H), 3.68 (d, 1H),3.0 (dd, 1H), 2.85 (dd, 1H), 1.29 (t, 3H) ppm; ¹³ C NMR (C₂ D₅ OD):δ169.6, 78.2, 77.9, 62.1, 38.3, 37.5, 13.9, ppm; Anal. Calcd for: C₇ H₁₂NO₂ Cl: C, 47.33; H, 6.81; N, 7.89; Cl, 19.96; Found: C, 47.42; H, 6.72;N, 7.70; Cl, 19.64.

EXAMPLE 5 ##STR11##

A mixture of 2.2 g of 4-aminobenzamidine dihydrochloride, 1.1 g ofsuccinic anhydride and 10 mL of DMF was stirred at ambient temperaturefor 15 min at which time 0.83 mL of pyridine was added all at once. Theresulting heterogeneous mixture was warmed to 100° C. and maintained at100° C. for 1.5 h. The disappearance of starting material was monitoredby an in-process check using HPLC Method B. After cooling to 0° C., asolution of 8 wt % concentrated HCl in acetone was added slowly whilemaintaining the temperature at 0° C. After the addition of additionalacetone, the resulting milky suspension was stirred for 30 min and thesolids were collected by vacuum filtration. The solids were then washedtwice each with aq 0.5M HCl followed by acetone and were dried in avacuum oven at 30°-40° C. to afford 2.4 g (88% molar yield) of the titlecompound. Acceptance testing was performed on the product using HPLCMethod B to determine the presence of the title product. λ_(max) (CH₃OH) 281 nm; (0.1% H₃ PO₄ in water) 203,270 nm; IR (KBr): 1736 (C═O),1674 (C--N, C═N), 1599 (C═N) cm⁻¹ ; DSC endotherms at 167° C. and 275°C.; ¹ H NMR: (d₆ -DMSO) δ12.15 (s, 1H), 10.55 (s, 1H), 9.23 (s, 2H), 9.0(s, 2H), 7.81 (s, 4H), 3.31 (s, 3H), 2.62 (m, 2H), 2.55 (m, 2H) ppm; ¹³C NMR: δ173.6, 171.0, 164.8, 144.3, 129.1, 121.2, 118.3, 31.2, 28.6 ppm;Anal calcd for C₁₁ H₁₄ N₃ O₃ Cl.0.25 H₂ O: C, 47.83, H, 5.29; N, 15.21;Cl, 12.84; Found: C, 47.68; H, 5.02; N, 15.23; Cl, 12.92.

EXAMPLE 6 ##STR12##

A mixture containing 0.95 g of4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid,monohydrochloride in 20 mL of DMAc was stirred at ambient temperatureuntil homogeneous at which time the solution was cooled to -10° C. Tothis cold solution was added 0.5 mL of isobutyl chloroformate followedby 0.43 mL of N-methylmorpholine while maintaining the batch temperaturebelow 0° C. After stirring this solution at -10° C. for 1 h a solutionof 0.63 g of ethyl 3S-amino-4-pentynoate monohydrochloride in 5 mL ofDMAc was added all at once followed by 0.38 mL of N-methylmorpholinewhile maintaining the batch temperature below 0° C. The temperature ofthe reaction mixture was adjusted to -5° C. and stirred at thistemperature for 3 h at which time the reaction mixture was warmed toambient temperature and the solvent was removed under reduced pressure.To the distillation residue was added 5 mL of 5% aq NaCl solution andthe resulting aqueous solution was stirred for 30 min at ambienttemperature. This solution was added dropwise over 30 min to 20 mL of a30% aq NaCl solution and the resulting milky suspension was stirred atambient temperature for 1 h. The suspension was vacuum filtered and thesolids were washed with 2×25 mL of 25% aq NaCl at which time they weredried in a vacuum oven at 50° C. until a Karl Fisher analysis (see U.S.Pharmacopeia XXIII, 1995, p. 1841 ) indicated that less than or equal to1 wt % of water was present. At this time the solids containing thetitle product, sodium chloride and reaction impurities were suspended inDMF (2.5 ml/g), stirred for 1 h and filtered to remove the NaCl. Thefiltrate was added to acetone (10 times the DMF volume), stirred for 2h, filtered and washed with additional acetone. The solids weresuspended in acetone (10 times DMF volume), refluxed for 1 h, cooled toambient temperature, filtered, washed with acetone (same as DMF volume)and dried in a vacuum oven overnight at 50° C. to afford 0.9 g of thetitle compound as an off-white powder (65% molar yield): [α](DMSO) -35.0at 589 nm, -131.7 at 365 nm; DSC: endotherms at 202.8° C. and 213.7° C.;IR: 1672¹ (C--N, C═N), 1721 (C═O), 2118 (C.tbd.C), 3352-3240 (NH, CN)cm.sup. -1 ; ¹ H NMR: (d₆ -DMSO) δ10.50 (s, 1H), 9.11 (br.s, 4H), 8.47(d, 1H), 7.80 (m, 4H), 4.87 (q.d, 1H), 4.07 (q, 2H), 3.22 (d, 1H),2.58-2.70 (m, 4H), 2.36-2.50 (m, 2H), 1.18 (t, 3H) ppm; ¹³ C NMR:δ171.1, 170.3, 169.1, 164.7, 144.2, 129.0, 121.1, 118.2, 82.8, 73.2,60.1, 40.0, 37.0, 31.4, 29.6, 13.9 ppm; Anal. Calcd for C₁₈ H₂₃ N₄ O₄Cl: C, 54.75; H, 5.87; N, 14.19; Cl, 8.98; Found: C, 54.39; H, 5.90; N,14.09; Cl, 9.24.

What we claim is:
 1. A process for the preparation of ethyl3S[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]-amino]-4-pentynoatehaving the following formula ##STR13## and the pharmaceuticallyacceptable acid addition salt thereof which comprises:(a) treating(trimethylsilyl)acetylene sequentially with n-butyllithium and4-formylmorpholine in the presence of an aprotic solvent followed byacid hydrolysis to give 3-(trimethylsilyl)-2-propynal; (b) treating3-(trimethylsilyl)-2-propynal, the product of step a, with lithiumbis(trimethylsilyl)amide in the presence of an aprotic solvent to giveN,3-bis(trimethylsilyl)-2-propyn-1-imine in situ, treatingN,3-bis(trimethylsilyl)-2-propyn-1-imine with lithium t-butyl acetatefollowed by hydrolytic cleavage to give (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate; (c) treating(±)1,1-dimethylethyl 3-amino-5-(trimethylsilyl)-4-pentynoate, theproduct of step b, with p-toluenesulfonic acid in the presence ofaprotic solvents to give (±)1,1-dimethylethyl3-amino-5-(trimethylsilyl)-4-pentynoate, mono p-toluenesulfonic acidsalt, treating the resulting salt with ethanol in the presence ofp-toluenesulfonic acid, followed by neutralization to give (±)ethyl3-amino-5-(trimethylsilyl)-4-pentynoate; (d) treating (±)ethyl3-amino-5-(trimethylsilyl)-4-pentynoate, the product of step c, with acatalytic amount of base in the presence of an alkanol solvent followedby a catalytic amount of acid to give the desilylated (±)ethyl3-amino-4-pentynoate in situ, treating (±)ethyl 3-amino-4-pentynoatewith (R)-(-)-mandelic acid in the presence of aprotic solvents to giveethyl 3S-amino-4-pentynoate compounded with αR-hydroxybenzeneaceticacid; (e) treating 4-aminobenzamidine dihydrochloride with succinicanhydride and pyridine in the presence of an aprotic solvent to give4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid,monohydrochloride; (f) treating ethyl 3S-amino-4-pentynoate compoundedwith αR-hydroxybenzeneacetic acid, the product of step d, with gaseoushydrochloric acid in the presence of an aprotic solvent to give ethyl3S-amino-4-pentynoate, monohydrochloride; and (g) treating4-[[4-(aminoiminomethyl)phenyl]amino]-4-oxobutanoic acid,monohydrochloride, the product of step e, with isobutyl chloroformateand N-methylmorpholine in the presence of an aprotic solvent followed byethyl 3S-amino-4-pentynoate, monohydrochloride, the product of step f,with N-methylmorpholine to give ethyl3S-[[4-[[4-(aminoiminomethyl)phenyl]amino]-1,4-dioxobutyl]amino]-4-pentynoate,monohydrochloride with the understanding that when a pharmaceuticallyacceptable acid addition salt other than hydrochloride is desired theethyl 3S-amino-4-pentynoate compounded with αR-hydroxybenzeneaceticacid, the product of step d, is treated with the appropriate acidcorresponding to the desired salt and with the further understandingthat the final salt of step e is identical to the final salt of step f.